Identification of Matriglycan by Dual Exoglycosidase Digestion of α-Dystroglycan

Matriglycan is a linear polysaccharide of alternating xylose and glucuronic acid units [-Xyl-α1,3-GlcA-β1,3]n that is uniquely synthesized on α-dystroglycan (α-DG) and is essential for neuromuscular function and brain development. It binds several extracellular matrix proteins that contain laminin-globular domains and is a receptor for Old World arenaviruses such as Lassa Fever virus. Monoclonal antibodies such as IIH6 are commonly used to detect matriglycan on α-DG. However, endogenous expression levels are not sufficient to detect and analyze matriglycan by mass spectrometry approaches. Thus, there is a growing need to independently confirm the presence of matriglycan on α-DG and possibly other proteins. We used an enzymatic approach to detect matriglycan, which involved digesting it with two thermophilic exoglycosidases: β-Glucuronidase from Thermotoga maritima and α-xylosidase from Sulfolobus solfataricus. This allowed us to identify and categorize matriglycan on α-DG by studying post-digestion changes in the molecular weight of α-DG using SDS-PAGE followed by western blotting with anti-matriglycan antibodies, anti-core α-DG antibodies, and/or laminin binding assay. In some tissues, matriglycan is capped by a sulfate group, which renders it resistant to digestion by these dual exoglycosidases. Thus, this method can be used to determine the capping status of matriglycan. To date, matriglycan has only been identified on vertebrate α-DG. We anticipate that this method will facilitate the discovery of matriglycan on α-DG in other species and possibly on other proteins. Key features • Analysis of endogenous matriglycan on dystroglycan from any animal tissue. • Matriglycan is digested using thermophilic enzymes, which require optimum thermophilic conditions. • Western blotting is used to assay the success and extent of digestion. • Freshly purified enzymes work best to digest matriglycan.

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.82811Matriglycan is a linear polysaccharide of alternating xylose and glucuronic acid units [-Xyl-α1,3-GlcA-β1,3]n that is uniquely synthesized on α-dystroglycan (α-DG) and is essential for neuromuscular function and brain development.It binds several extracellular matrix proteins that contain laminin-globular domains and is a receptor for Old World arenaviruses such as Lassa Fever virus.Monoclonal antibodies such as IIH6 are commonly used to detect matriglycan on α-DG.However, endogenous expression levels are not sufficient to detect and analyze matriglycan by mass spectrometry approaches.Thus, there is a growing need to independently confirm the presence of matriglycan on α-DG and possibly other proteins.We used an enzymatic approach to detect matriglycan, which involved digesting it with two thermophilic exoglycosidases: β-Glucuronidase from Thermotoga maritima and αxylosidase from Sulfolobus solfataricus.This allowed us to identify and categorize matriglycan on α-DG by studying post-digestion changes in the molecular weight of α-DG using SDS-PAGE followed by western blotting with antimatriglycan antibodies, anti-core α-DG antibodies, and/or laminin binding assay.In some tissues, matriglycan is capped by a sulfate group, which renders it resistant to digestion by these dual exoglycosidases.Thus, this method can be used to determine the capping status of matriglycan.To date, matriglycan has only been identified on vertebrate α-DG.We anticipate that this method will facilitate the discovery of matriglycan on α-DG in other species and possibly on other proteins.

Background
α-Dystroglycan (α-DG) is an extensively glycosylated and widely expressed transmembrane protein that functions as an extracellular matrix receptor.It is modified with several types of O-and N-glycans.The glycans on α-DG enable it to interact with the extracellular molecules and maintain cell stability and integrity.The most widely studied post-translational modification of α-DG is the addition of the O-mannosylated glycan called core M3.This glycan is defined as a phosphorylated O-mannosyl trisaccharide [GalNAcβ1-3GlcNAcβ1-4(phosphate-6)Man-O-Ser] comprised of a mannose that is extended by the sequential addition of an N-acetylglucosamine and an N-acetyl galactosamine (Yoshida-Moriguchi and Campbell, 2015).The C6 hydroxyl group of the mannose of core M3 is also phosphorylated by a kinase (Yoshida-Moriguchi et al., 2013).Core M3 is further elongated, from the Nacetylgalactosamine, by a glycan modification that terminates in the repeating disaccharide of xylose (Xyl) and glucuronic acid [-Xyl-α1,3-GlcA-β1,3-]n called matriglycan.Notably, over 18 genes are involved in generating the final core M3 structure (Walimbe et al., 2020).Matriglycan acts as a scaffold for laminin-G domain containing proteins (e.g., laminin, agrin, perlecan, and neurexin) in the extracellular matrix.Loss-of-function mutations in any of the 18 genes involved in matriglycan synthesis lead to different types of muscular dystrophies, including severe forms such as Walker-Warburg syndrome.Owing to the complexity of the final core M3 structure terminating in matriglycan and its low expression levels in cells, it has never been analyzed using mass spectrometry.Therefore, to study and to confirm the presence of matriglycan on dystroglycan in various animal tissues, mutants, and patient samples, we use an enzymatic approach.We identified two exoglycosidases that together can hydrolyze matriglycan in native tissues: β-glucuronidase (Bgus) from Thermotoga maritima (Salleh et al., 2006)  .In conjunction with mass spectrometry and chemically synthesized matriglycan, we previously demonstrated that these enzymes sequentially remove sugars from the non-reducing end and that Bgus and Xyls specifically cleave the β1,3-linked GlcA and α1,3-linked Xyl, respectively (Briggs et al., 2016).Given that neither enzyme has detectable endoglycosidase activity (Briggs et al., 2016), they enable us to probe the matriglycosylation status of native α-DG.Matriglycan from brain was found to be capped by a 3-O-sulfation of its terminal GlcA, a reaction catalyzed by HNK-1 sulfotransferase.The sulfate cap prevents further elongation of matriglycan and also makes it resistant to digestion by the dual exoglycosidases.It has been previously demonstrated that the matriglycan on α-DG isolated from brain is capped by sulfation and becomes susceptible to digestion by the exoglycosidases after digesting it with a sulfatase (Sheikh et al., 2020).Therefore, this method enables us to probe the tissuedependent matriglycosylation status of α-DG.To date, matriglycan has been found only on α-DG, although a report suggests it could be present on glypican-4 (Inamori et al., 2016).This digestion protocol is expected to be useful in confirming the presence of matriglycan on other proteins such as glypican and may also aid in discovering new proteins modified with matriglycan.Also, matriglycan has been found only in vertebrate α-DG, e.g., in human, mouse, and zebrafish (Praissman et al., 2016;Liu et al., 2020), and this protocol can be used to determine whether matriglycan exists in other species.Such analyses have the potential to broaden the use of other species as a model organism for the study of muscular dystrophies arising from changes in matriglycosylation of α-DG.Although matriglycan can be detected using antibodies, those currently available sometimes detect nonspecific bands; therefore, confirming the presence of matriglycan independently will be important, especially when working with new proteins or organisms.In summary, the technique described here is expected to be useful for definitively detecting matriglycan (in contrast to antibodies) on new proteins and in new organisms.One limitation of using thermophilic enzymes for digestion is that they require high temperatures and acidic pH to function, which could be detrimental to some substrate proteins.We have found α-DG to remain remarkably stable at high temperatures and acidic pH, making the use of the dual thermophilic exoglycosidases feasible (Briggs et al., 2016).

D. Digestion of α-Dystroglycan (α-DG)
1. Use wheat germ agglutinin (WGA) agarose to enrich α-DG from animal tissues, as follows: a. Harvest 1 g of skeletal muscle from C57BL/6J mice.Pour liquid nitrogen into a mortar and pestle and crush the muscle in it.Note: Take muscles from the legs after euthanizing mice by cervical dislocation.Briefly, remove the skin by making a small cut in the mid-dorsal or ventral region and peel it off the mouse; then, using scissors and forceps, take all muscles from the legs including quadriceps, tibialis anterior, and soleus, until bone is visible.
b. Transfer powdered muscle into a vial and allow to warm for 3-5 min, either on ice or in a cold room.Note: Vial should be able to withstand cold liquid nitrogen.We use plastic liquid scintillation vials.c.Add 10 mL of solubilization buffer and mince the tissue in a polytron homogenizer for 15 s at speed 4. Repeat this three times.d.Transfer solution to a 50 mL glass homogenizer and macerate tissue by moving the plunger in and out of the solution at least 10 times.Note: For macerating tissue with an automated overhead stirrer (instead of doing it by hand), use speed 4. e. Transfer homogenized material to a 50 mL Falcon tube and incubate on a rotator for 1 h at 4 °C.f.Spin the material for 30 min at 20,000× g at 4 °C.Transfer supernatant to new tube.g.Equilibrate 500 μL of WGA beads with 5 mL of solubilization buffer.Spin down beads at 1,500× g for 5 min.Remove buffer.h.Combine material obtained in step D1e with the equilibrated WGA beads and incubate for 16 h on rotator at 4 °C.i. Spin down beads at 1,500× g for 5 min at 4 °C and save the supernatant as WGA-void fraction.j.Wash WGA beads with 5 mL of WGA wash buffer by incubating for 5 min on a rotator and then spinning beads at 1,000× g for 10 min at 4 °C.Repeat this wash step four times.k. α-DG is now on the WGA beads.Add 1 mL of WGA elution buffer to elute α-DG from 500 μL of WGA beads and incubate at 4 °C for 1 h on a rotator.l.Spin down beads at 1,500× g for 5 min at 4 °C.α-DG is now in the supernatant or eluate.

E. SDS-PAGE and western blotting
We analyze the progression and effectiveness of digestion by running the samples in 3%-15% SDS-PAGE gradient gels and performing immunoblotting on PVDF membranes, using antibodies against matriglycan (IIH6, 1:100), against core α-DG (AF6868 1:200) (Figure 2), and/or laminin overlay and solid phase laminin binding

Data analysis
Upon successful digestion, clear shifts in the molecular weights of α-DG and matriglycan are observed.We also measure changes in laminin binding by performing laminin overlay and solid phase binding analysis (Walimbe et al., 2020, Figure 5D and 5E).Blots are scanned using a Li-Cor Odyssey imaging system and fluorescence detection, and images are analyzed using the Image Studio software.A detailed description of data analysis was provided previously (Briggs et al., 2016, Supplementary figure 1).

Validation of protocol
We have routinely used this method to identify matriglycan and found it to be robust and reproducible (Okuma et al., 2023;Briggs et al., 2016;Sheikh et al., 2020;Walimbe et al., 2020).Additional data validating our method is provided in Figure 1 and Figure 2.

General notes
We have found that these enzymes are most effective when freshly purified.

Troubleshooting
If the enzymes are 4-5 months old and do not seem to work, it is best to purify fresh samples.

9 Published 11 Published: Sep 20, 2023 Figure 1 .
Figure 1.Purification of Bgus and Xyls. A. Column-purified Bgus is shown here in Eluates 1 and 2 and runs at ~66 kD in a 3%-15% gradient SDS-PAGE gel stained with Coomassie blue.B. Column-purified Xyls is shown here in Eluates 1 and 2 and runs at ~80 kD in a 3%-15% gradient SDS-PAGE gel stained with Coomassie blue.